Although the catalytic (C) subunit of cAMP-dependent protein kinase is N-myristylated, it is a soluble protein, and no physiological role has been identified for its myristyl moiety. To determine whether the interaction of the two regulatory (R) subunit isoforms (R I and R II ) with the N-myristylated C subunit affects its ability to target membranes, the effect of N-myristylation and the R I and R II subunit isoforms on C subunit binding to phosphatidylcholine͞ phosphatidylserine liposomes was examined. Only the combination of N-myristylation and R II subunit interaction produced a dramatic increase in the rate of liposomal binding. To assess whether the R II subunit also increased the conformational flexibility of the C subunit N terminus, the effect of N-myristylation and the R I and R II subunits on the rotational freedom of the C subunit N terminus was measured. Specifically, fluorescein maleimide was conjugated to Cys-16 in the N-terminal domain of a K16C mutant of the C subunit, and the time-resolved emission anisotropy was determined. The interaction of the R II subunit, but not the R I subunit, significantly increased the backbone flexibility around the site of mutation and labeling, strongly suggesting that R II subunit binding to the myristylated C subunit induced a unique conformation of the C subunit that is associated with an increase in both the N-terminal flexibility and the exposure of the N-myristate. R II subunit thus appears to serve as an intermolecular switch that disrupts of the link between the N-terminal and core catalytic domains of the C subunit to expose the N-myristate and poise the holoenzyme for interaction with membranes. C yclic AMP-dependent protein kinase (cAPK) exists in nearly all eukaryotic cells and plays a critical role in the regulation of cellular growth, metabolism, and homeostasis by catalyzing the phosphorylation of a variety of proteins (1-3). The holoenzyme configuration of cAPK comprises two catalytic (C) subunits and a cAMP-binding regulatory (R) subunit homodimer. Three mammalian forms of the C subunit have been identified (C ␣ , C ␤1,2,3 , and C ␥ ) (1-3). With the exception of some of the novel C ␤ splice variants, all the mammalian isoforms are myristylated (4, 5). Two pharmacologically and structurally distinct types of the R subunits, R I and R II , are known, along with ␣ and ␤ isoforms of each (1-3). Tissue-specific membrane targeting of each type has been observed (1-3) and appears to be mediated by specific membrane anchoring proteins (A-kinase anchoring proteins) that bind to the dimerization domain of the R subunit but not to the C subunit (6).Because protein N-myristylation is often part of a membrane targeting signal that permanently or reversibly steers proteins to membranes (7), it is perplexing that the C subunit is thought to lack a membrane targeting signal and that membrane binding of the holoenzyme results solely from A-kinase anchoring protein interaction with the nonmyristylated R subunits. The x-ray structure of the mammalian C ␣ subunit su...

To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.